Plant Pathogenic Fungus Botrytis Cinerea Research Articles

The F-actin capping protein is required for hyphal growth and full virulence but is dispensable for septum formation in Botrytis cinerea

Filamentous (F-) actin is an integral part of the cytoskeleton allowing for cell growth, intracellular motility, and cytokinesis of eukaryotic cells. Its assembly from G-actin monomers and its disassembly are tightly regulated processes involving a number of actin-binding proteins (ABPs) such as F-actin nucleators and cross-linking proteins. F-actin capping protein (CP) is an alpha/beta heterodimer known from yeast and higher eukaryotes to bind to the fast growing ends of the actin filaments stabilizing them. In this study, we identified the orthologs of the two CP subunits, named BcCPA1 and BcCPB1, in the plant pathogenic fungus Botrytis cinerea and showed that the two proteins physically interact in a yeast two-hybrid approach. GFP-BcCPA1 fusion proteins were functional and localized to the assumed sites of F-actin accumulation, i.e. to the hyphal tips and the sites of actin ring formation. Deletion of bccpa1 had a profound effect on hyphal growth, morphogenesis, and virulence indicating the importance of F-actin capping for an intact actin cytoskeleton. As polarized growth – unlike septum formation – is impaired in the mutants, it can be concluded that the organization and/or localization of actin patches and cables are disturbed rather than the functionality of the actin rings.

A novel Zn2 Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea.

D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2 Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

Novel hypovirulence-associated RNA mycovirus in the plant-pathogenic fungus Botrytis cinerea: molecular and biological characterization.

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A -1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.

Around the Fungal Clock: Recent Advances in the Molecular Study of Circadian Clocks in Neurospora and Other Fungi.

Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages.

Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches

Targeted gene inactivation is extensively used in the plant pathogenic fungus Botrytis cinerea for gene function analysis while strategies involving the expression of reporter genes have been rarely used due to the lack of appropriate expression vectors. Hence, an approach was initiated to establish an expression system for B.cinerea possessing the following features: (i) the targeted integration of constructs at defined gene loci which are dispensable under standard growth conditions, (ii) the use of promoter and terminator sequences allowing optimal gene expression, (iii) the use of codon-optimized reporter genes (Leroch et al., 2011), (iv) the use of multiple selection markers, and (v) the incorporation of a highly efficient cloning system. A set of basic vectors was generated by yeast recombinational cloning permitting a variety of protein fusions. The successful application of the expression system for labeling F-actin, the cytosol, the nuclei, the membrane, the ER and the peroxisomes was demonstrated. In addition, cloning vectors for bimolecular fluorescence complementation (BiFC) analyses for studying protein–protein interactions in situ were generated by splitting the codon-optimized gfp. The functionality of the constructed BiFC vectors was validated by demonstrating the interaction of the two white collar-like transcription factors BcWCL1 and BcWCL2 in the nuclei of growing B. cinerea hyphae.

Host-targeting-motif Harbored Secretary Proteins in Genome of Plant Pathogenic Fungus Botrytis cinerea

According to our previous study, saprophytic fungi Botrytis cinerea contained 579 predicted secretary proteins. Among them, we found that 122 of these proteins contained the highly conserved pathogenic-related host-targeting-motif RxLx within 100 residues adjacent to the signal peptide cleavage site. According to PEDNAT and COG of the GenBank database, the functions of this motif containing proteins included metabolism modification and cell secretion. We blasted them in GenBank and found 47.54% had highly conserved homologues in other species, among them 74.1% had putative functional domains. This suggests these proteins are presumably ancient and vertically transmitted within the species. Many of these domains belonged to proteins which played roles in the pathogenic process of other kinds of pathogens and some had already been proved to be pathogenic secretary proteins of Botrytis cinerea. So we postulated that proteins contained host-targeting-motif RxLx were candidates participating in the pathogenesis of Botrytis cinerea.

The d-galacturonic acid catabolic pathway in Botrytis cinerea

d-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving d-galacturonate reductase, l-galactonate dehydratase, and 2-keto-3-deoxy-l-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire d-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on d-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the d-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.

Deleterious Effect of the Q o Inhibitor Compound Resistance-Conferring Mutation G143A in the Intron-Containing Cytochrome b Gene and Mechanisms for Bypassing It

The mutation G143A in the inhibitor binding site of cytochrome b confers a high level of resistance to fungicides targeting the bc(1) complex. The mutation, reported in many plant-pathogenic fungi, has not evolved in fungi that harbor an intron immediately after the codon for G143 in the cytochrome b gene, intron bi2. Using Saccharomyces cerevisiae as a model organism, we show here that a codon change from GGT to GCT, which replaces glycine 143 with alanine, hinders the splicing of bi2 by altering the exon/intron structure needed for efficient intron excision. This lowers the levels of cytochrome b and respiratory growth. We then investigated possible bypass mechanisms that would restore the respiratory fitness of a resistant mutant. Secondary mutations in the mitochondrial genome were found, including a point mutation in bi2 restoring the correct exon/intron structure and the deletion of intron bi2. We also found that overexpression of nuclear genes MRS2 and MRS3, encoding mitochondrial metal ion carriers, partially restores the respiratory growth of the G143A mutant. Interestingly, the MRS3 gene from the plant-pathogenic fungus Botrytis cinerea, overexpressed in an S. cerevisiae G143A mutant, had a similar compensatory effect. These bypass mechanisms identified in yeast could potentially arise in pathogenic fungi.

Investigation on the infection mechanism of the fungus Clonostachys rosea against nematodes using the green fluorescent protein

The fungus Clonostachys rosea (syn. Gliocladium roseum) is a potential biocontrol agent. It can suppress the sporulation of the plant pathogenic fungus Botrytis cinerea and kill pathogenic nematodes, but the process of nematode pathogenesis is poorly understood. To help understand the underlying mechanism, we constructed recombinant strains containing a plasmid with both the enhanced green fluorescent protein gene egfp and the hygromycin resistance gene hph. Expression of the green fluorescent protein (GFP) was monitored using fluorescence microscopy. Our observations reveal that the pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. These are the first findings on the infection process of the fungal pathogen marked with GFP, and the developed method can become an important tool for studying the molecular mechanisms of nematode infection by C. rosea.

Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad‐spectrum antibiotic 2,4‐diacetylphloroglucinol

The genetic and biochemical basis of defence mechanisms in plant pathogenic fungi against antifungal compounds produced by antagonistic microorganisms is largely unknown. The results of this study show that both degradative and non-degradative defence mechanisms enable the plant pathogenic fungus Botrytis cinerea to resist the broad-spectrum, phenolic antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The efflux pump BcAtrB provides the first line of defence for B. cinerea, preventing accumulation of 2,4-DAPG in the cell to toxic concentrations, whereas the extracellular laccase BcLCC2 mediates, via conversion of tannic acid, subsequent degradation of 2,4-DAPG. Expression of BcatrB is induced by 2,4-DAPG and efflux gives B. cinerea sufficient time to more effectively initiate the process of BcLCC2-mediated antibiotic degradation. This is supported by the observations that the BcatrB mutant is significantly more sensitive to 2,4-DAPG than its parental strain, and is substantially less effective in 2,4-DAPG degradation. The results of this study further showed that BcLCC2 itself is not able to degrade 2,4-DAPG, but requires tannic acid as a mediator for 2,4-DAPG degradation. To our knowledge, this is the first time that the laccase-mediator system is shown to play a role in the detoxification of a broad-spectrum antibiotic compound from bacterial origin. We postulate that yet unknown constituents present in tannic acid act as substrate(s) of BcLCC2, thereby generating radicals that mediate 2,4-DAPG degradation.

Phytotoxic Nep1‐like proteins from the necrotrophic fungus Botrytis cinerea associate with membranes and the nucleus of plant cells

Nep1-like proteins (NLPs), produced by an array of unrelated microorganisms, are phytotoxic for dicotyledonous plant cells but their mode of action has not yet been established. Two paralogous NLPs from the necrotrophic plant pathogenic fungus Botrytis cinerea were characterized, designated BcNEP1 and BcNEP2. Both proteins were produced in the heterologous host Pichia pastoris and purified to homogeneity. The localization of fluorescently labelled proteins was studied and mechanisms of cell death were investigated in protoplasts and suspension cells. Purified BcNEP1 and BcNEP2 caused necrosis in all dicotyledonous plant species tested, but not in monocotyledons. A synthetic heptapeptide comprising a sequence (GHRHDWE) that is conserved in all NLPs did not cause symptoms and was unable to interfere with necrosis induction by BcNEP1 and BcNEP2 proteins. Fluorescently labelled BcNEP1 and BcNEP2 proteins were associated with plasma membranes and the nuclear envelope, as well as in the nucleolus of responding plant cells. A strong hydrogen peroxide (H(2)O(2)) accumulation was observed in chloroplasts. The death process was characterized by TUNEL assays as apoptosis, necrosis or intermediate forms of both. BcNEP1- and BcNEP2-induced cell death execution could not be abolished by specific inhibitors. These results provide further information on mechanisms of NLP-inflicted cell death.

A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis

The phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16-dihydroxypalmitic or 2,15,16-trihydroxypalmitic acid, which is O-glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short-chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non-haem diiron reductases and is required for alpha-hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co-inoculation with wild-type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu–Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.

Modeling Spatial Characteristics in the Biological Control of Fungi at Leaf Scale: Competitive Substrate Colonization by Botrytis cinerea and the Saprophytic Antagonist Ulocladium atrum

ABSTRACT A spatially explicit model describing saprophytic colonization of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum was constructed. Both fungi explore the leaf and utilize the resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid cells. Fungal competition within grid cells is modeled using Lotka-Volterra equations. Spatial expansion into neighboring grid cells is assumed proportional to the mycelial density gradient between donor and receptor cell. Established fungal biomass is immobile. Radial growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to determine parameters describing the spatial dynamics of the fungi. At temperatures from 5 to 25 degrees C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical biological control, the slower colonization of space by U. atrum thus needs to be compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation results confirm the importance of spatial expansion to the outcome of the competitive interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further emphasized the importance of a uniform high density cover of vital U. atrum conidia on target leaves.

Antifungal activity of limonoids from Khaya ivorensis

Chemical investigation of the diethyl ether extract of the stem bark of Khaya ivorensis A Chev (Meliaceae) afforded ten limonoids of angolensates, ring D-opened limonoids and mexicanolides. The structures of the limonoids isolated were determined by comparison of their (1)H and (13)C NMR data with those reported in the literature. These compounds were evaluated for their antifungal activity against the plant pathogenic fungus Botrytis cinerea Pers. Methyl 6-hydroxyangolensate and 3,7-dideacetylkhivorin were also tested for their antifungal and antibacterial activities on several fungal and bacterial species. Methyl angolensate and 1,3,7-trideacetylkhivorin displayed the highest antifungal activity against B. cinerea, with respectively 62.8 and 64.0% mycelial growth inhibition at 1000 mg litre(-1), and 73.3 and 68.6% mycelial growth inhibition at 1500 mg litre(-1). 3,7-Dideacetylkhivorin showed stronger antifungal and antibacterial activities than methyl 6-hydroxyangolensate against all of the test fungi and bacteria except Penicillium expansum Link. This is the first report on the antifungal and antibacterial effects of these limonoids. Structure-antifungal activity relationships of the limonoids isolated are discussed.

Functional Analysis of ABC Transporter Genes FromBotrytis cinereaIdentifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA-BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.

Fungal ABC transporters and microbial interactions in natural environments.

In natural environments, microorganisms are exposed to a wide variety of antibiotic compounds produced by competing organisms. Target organisms have evolved various mechanisms of natural resistance to these metabolites. In this study, the role of ATP-binding cassette (ABC) transporters in interactions between the plant-pathogenic fungus Botrytis cinerea and antibiotic-producing Pseudomonas bacteria was investigated in detail. We discovered that 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and phenazine-1-carboxamide (PCN), broad-spectrum antibiotics produced by Pseudomonas spp., induced expression of several ABC transporter genes in B. cinerea. Phenazines strongly induced expression of BcatrB, and deltaBcatrB mutants were significantly more sensitive to these antibiotics than their parental strain. Treatment of B. cinerea germlings with PCN strongly affected the accumulation of [14C]fludioxonil, a phenylpyrrole fungicide known to be transported by BcatrB, indicating that phenazines also are transported by BcatrB. Pseudomonas strains producing phenazines displayed a stronger antagonistic activity in vitro toward ABcatrB mutants than to the parental B. cinerea strain. On tomato leaves, phenazine-producing Pseudomonas strains were significantly more effective in reducing gray mold symptoms incited by a ABcatrB mutant than by the parental strain. We conclude that the ABC transporter BcatrB provides protection to B. cinerea in phenazine-mediated interactions with Pseudomonas spp. Collectively, these results indicate that fungal ABC transporters can play an important role in antibiotic-mediated interactions between bacteria and fungi in plant-associated environments. The implications of these findings for the implementation and sustainability of crop protection by antagonistic microorganisms are discussed.

Detoxification of terpinolene by plant pathogenic fungus Botrytis cinerea.

Detoxification of an antifungal monoterpene terpinolene (1) by the plant pathogenic fungus Botrytis cinerea afforded hydroxlyated metabolites 2,3-dihydro-3beta,6beta-dihydroxy-terpinolene (2) (39%) and 2,3-dihydro-1alpha,3alpha-dihydroxy-terpinolene (3) (20%), respectively. Terpinolene showed good levels of antifungal activity while both the metabolites were inactive against another plant pathogenic fungus Cladosporium herbarun.

Competitive substrate colonisation by Botrytis cinerea and Ulocladium atrum in relation to biological control of B. cinerea in cyclamen

The dynamics of competitive colonisation of necrotic cyclamen tissue by the plant pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum were studied immuno-histologically, while sporulation was studied macroscopically. The effect of different time intervals between inoculation of both fungi on resource capture by each species was explored. Colonisation and sporulation were used as indicators for competitive resource capture and the effectiveness of biological control of B. cinerea, using U. atrum. Mycelial biomass and sporulation showed logistical time courses in both species, in monocultures as well as in mixed cultures. Final colonisation and sporulation levels were lowered by competition, indicating competitive resource capture. Analysis of the extent to which sporulation of either fungus could be reduced by co-inoculation with the other fungus at different times, showed that B. cinerea can be completely excluded by ‘early’ pre-inoculation with U. atrum, but not vice versa, indicating that U. atrum can exploit resources in the leaf that are not accessible to B. cinerea. A model of competitive substrate colonisation and resource capture was developed on the basis of the experimental results. Model results confirm that competition for resources provides a sufficient biological explanation for the dynamic interactions between the fungi. The model provides a tool to optimise dose and timing of U. atrum applications providing effective biological control of B. cinerea.